Research Abstract The goal of this project is to elucidate the enzymatic pathways that underlie the biosynthesis of lipids that are produced by human meibomian glands (HMG) and are collectively known as meibum. Meibum is a critically important part of the tear film (TF) that, among other functions, protects the ocular surface from desiccating. Meibum is formed predominantly from a unique mixture of very, and extremely, long chain complex lipids. Our previous projects were devoted to biochemical and biophysical characterization of meibomian lipids (ML). Now we propose to investigate the biosynthetic pathways that lead to the formation of meibum in HMG. The following three lines of research are proposed: (I). Despite recent progress in the characterization of the lipidomes of meibum and TF, there is no clear understanding of the biosynthetic path- ways involved in the biosynthesis of meibum. The unusual and, often, unique qualities of ML imply their in situ and/or de novo origination, which, in turn, requires proper molecular mechanisms to be implemented in HMG. We will study the levels of expression, and map, major lipid-metabolizing enzymes and related proteins, which, based on our preliminary studies, are expected to be present in HMG. This will be achieved by using a combination of approaches, such as microarray RNA analyses, immunohistochemistry/immunocytochemistry, in-situ hybridization, proteomics, and lipidomics. (II). Recent reports indicate that high total blood lipid levels, including cholesterol and TAG, are correlated with higher incidence and/or severity of MG dysfunction. Also, a positive impact of ?3 fatty acids (?3FA) supplements on the ocular surface was reported for humans and mice. Yet, the reasons and molecular mechanisms of these effects are unknown. We will determine the balance between the in situ/de novo biosynthesis of ML and the levels and composition of blood lipids. We will test if 1) enhanced intake of pharmaceutical doses of ?3FA, and (2) use of statins, can have a positive impact on the biosynthesis, composition and properties of meibum. (III). Recently, we reported that: (1) up to 20% of normal meibum is formed of non-lipid components; (2) there are considerable differences between normal and DE meibum: the latter is enriched with non-lipid material, and forms lipid layers that are less structurally stable than those formed of healthy meibum. Based on those findings, we propose to assess the non-lipid content of mei- bum using: (a) metabolomic approaches based on HPLC-MS, GC-MS, and 2D-DESI MS; (b) Western blot with specific antibodies against major meibum and TF proteins; (c) histochemical approaches targeting proteins and polysaccharides; (d) various biophysical methods, such as hot stage polarized light microscopy, microcalori- metry, Brewster angle microscopy, to study rheological and melting characteristics of meibum and tear film lipids. We believe that our work will provide innovative information that may lead to new concepts in medical treatment of MG-related diseases and conditions, and may establish a link between them and dyslipidemia.